The pharmacokinetics, pharmacodistribution, solubility, stability, enhancement of effector function and receptor binding of protein therapeutics can be significantly influenced by the carbohydrate moiety of glycosylated proteins. In addition, many biologically active peptides and proteins have limited solubility, or become aggregated during recombinant productions, requiring complex solubilization and refolding procedures. Furthermore, protein and peptide therapeutics with molecular weights lower than 60 kilodaltons (kD) often suffer from short half-lives due to renal clearance.
Current strategies employed to extend serum half-life of protein therapeutics primarily fall within two general categories: 1) utilization of FcRn-mediated recycling and 2) increase of hydrodynamic volume. Specific approaches which have been described include conjugation, binding, or fusion to FcRn-binding proteins or domains (Fc, albumin) for the former strategy, and multimerization, chemical coupling to polymers or carbohydrates (such as PEG, Colominic acid, or Hydroxyethyl starch), incorporation of N-glycosylation sites for the latter. However, the production of Fc-fusion proteins is a time-consuming, inefficient, and expensive process that requires additional manufacturing steps and often complex purification procedures. In addition, chemical coupling strategies, PEGylation being the most widely used, result in significant increases in production costs due to the addition of conjugation and purification steps and reduced overall yields. Recently, other recombinant PEG mimetics produced through fusion of a long, flexible polypeptide sequence, such as those described in U.S. 2010/0239554 A1, have also been described. Although this technology circumvents the additional conjugation step, the added peptide sequence, being non-endogenous, has the potential for immunogenicity.
Mucin proteins and mucin-domains of proteins contain a high degree of glycosylation which structurally allows mucin proteins and other polypeptides comprising mucin domains to behave as stiffened random coils. This stiffened random coiled structure in combination with the hydrophilic branched hydrophilic carbohydrates that make up the heavily glycosylated mucin domains is particularly useful in for increasing the hydrodynamic radius of the active protein beyond what would be expected based on the molecular weight of the expressed protein. Also because of the high level of glycosylation, addition of a mucin domain has the potential to modify the physicochemical properties of a protein such as charge, solubility and viscoelastic properties of concentrated solutions of the active protein.
The fusion protein compositions and methods of the present invention improve the biological, pharmacological, safety, and/or pharmaceutical properties of an active protein.